Methane (CH4) is the second most prevalent greenhouse gas emitted in the United States, accounting for about 9% of all United States greenhouse gas emissions from human activities in 2012. Methane is emitted by natural sources such as wetlands, as well as human activities such as natural gas systems and agriculture. Additionally, methane is much more efficient at trapping radiation than carbon dioxide (CO2), the most prevalent greenhouse gas emitted in the United States. In fact, pound for pound, the comparative impact of methane on climate change is over 20 times greater than carbon dioxide over a 100-year period (United States Environmental Protection Agency).
Methanotrophs are microorganisms that oxidize methane to carbon dioxide and water via the intermediates methanol, formaldehyde, and formate. They play a major role in reducing the methane released from natural environments such as rice paddies, landfills, bogs, and swamps, where methane production is relatively high. As such, methanotrophs have attracted attention from environmental scientists for their potential in bioremediation efforts, namely the reduction of atmospheric methane levels and the mitigation of the effects of global warming.
Aerobic methanotrophs overcome the high activation energy (439 kJ/mol) (Trotsenko, Adv Appl Microbiol, 63: 183-229, 2008) required to break the C—H bond of methane by using oxygen as a highly reactive co-substrate for the initial attack, in a reaction catalysed by methane monooxygenase. In particular, methane monooxygenase uses two reducing equivalents from NAD(P)H to split the O—O bond of O2, whereby one atom is reduced to water and the second atom is incorporated into the substrate to yield methanol: CH4+NAD(P)H+H++O2→CH3OH+NAD(P)++H2O. However, gaseous substrates comprising methane and oxygen are highly combustible, rendering industrial-scale growth of aerobic methanotrophs problematic, if not prohibitively dangerous.
Prior to the discovery of Candidatus Methylomirabilis oxyfera (Ettwig, Nature, 464: 543-548, 2010), it was believed that that anaerobic oxidation of methane by a single microorganism was biologically impossible (Wu, Biochem Soc Trans, 39: 243-248, 2011). Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in syntrophic consortia of methanotrophic archaea and reducing bacteria, M. oxyfera produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas (Raghoebarsing, Nature, 440: 918-921, 2006; Ettwig, Appl Environ Microbiol, 75: 3656-3662, 2009; Hu, Environ Microbiol Rep, 1: 377-384, 2009; Ettwig, Nature, 464: 543-548, 2010; Luesken, Environ Microbiol, 14: 1024-1034, 2012). The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane monooxygenase (Ettwig, Nature, 464: 543-548, 2010).
Although M. oxyfera does not require a combustible gaseous substrate containing methane and oxygen like aerobic methanotrophs, M. oxyfera has not yet been isolated in pure culture, grown at scale, or shown to produce any commercially valuable products. Accordingly, there remains a strong need for microorganisms and methods capable of converting methane to useful products, such as alcohols or acids.